The interaction between cholesterol and human serum albumin has been investigated. The basic binding interaction was studied by UVabsorption and fluorescence spectroscopy. From spectral analysis cholesterol showed a strong ability to quench the intrinsic fluorescence of HSA through a static quenching mechanism. The binding constant (k) is estimated to be K=0.214 × 104 M-1 at 293 K. FTIR spectroscopy with Fourier self-deconvolution technique was used to determine the protein secondary structure and cholesterol binding mechanisms. The observed spectral changes indicate a higher percentage of H-bonding between cholesterol and a-helix (secondary structure motif in HSA) compared to the percentage of H-bonding between cholesterol and b-sheets (secondary structure motif in HSA)